Our long term goals are to understand the means by which Borrelia burgdorferi (Bb) disseminates and subsequently persists in its hosts. Basic information about the phenomenology of Bb interaction with host cells and tissues is needed so that potential mechanisms of dissemination and persistence can be more precisely formulated. Freeze-fracture electron microscopy can reveal in detail the structure of adjacent membranes involved in a cell-cell interaction; we will continue to use free-fracture as a tool to describe Bb binding to cells of tick midgut and central ganglion, rat synovium, and Peromyscus bladder as well as to cultured cell lines of human endothelial and epithelial origin. These studies will reveal whether there is a specialized Bb attachment structure, whether Bb membrane reorganization occurs in conjunction with host cell binding, and will detect in vivo alteration of Bb outer membrane protein content. Our finding that Bb, like Treponema pallidum, can invade endothelial intercellular junctions represents a potential dissemination mechanism; we will define the relationship of invasion of endothelial and epithelial intercellular junctions by Bb to virulence. We will also clarify whether the endothelial cell membrane bound Bb which we have observed are intracellular, and if so, whether their fate is transcytosis or persistence. Basic information regarding the surface composition of Bb in vivo as well as in vitro is also needed to establish a framework from which molecules relevant to dissemination and persistence can ultimately be identified. Several lines of evidence suggest that Bb has altered surface properties in vivo; borrelial persistence in a wide range of environments may reflect specific changes in Bb surface composition made in response to specific tissue and host microenvironments. The small numbers of Bb present in vertebrate infection have made this a difficult matter to address experimentally. We have designed a genetic system, based on in vitro gene fusions between Bb signal sequences and alkaline phosphatase, which has the power to identify Bb genes which encode virulence related Bb proteins. We will be able to learn which of these genes are silent in vitro but are expressed in certain tissues and hosts. Because we believe that minor molar constituents of the Bb surface may play a role in pathogenesis, and also to create a foundation for evaluating the cellular location of proteins which will be derived by the use of our genetic system, we will define surface proteins of in vitro grown Bb by preparative immunoaffinity purification and by phase partitioning with Triton X-114.